Methods for detection of ataxia telangiectasia mutations

ABSTRACT

The present invention is directed to a method of screening large, complex, polyexonic eukaryotic genes such as the ATM gene for mutations and polymorphisms by an improved version of single strand conformation polymorphism (SSCP) electrophoresis that allows electrophoresis of two or three amplified segments in a single lane. The present invention also is directed to new mutations and polymorphisms in the ATM gene that are useful in performing more accurate screening of human DNA samples for mutations and in distinguishing mutations from polymorphisms, thereby improving the efficiency of automated screening methods.

RELATED APPLICATIONS

[0001] This is a divisional of U.S. patent application Ser. No.09/360,416, filed on Jul. 23, 1999.

GOVERNMENT RIGHTS

[0002] This invention was made with Government support under Grant No.DEFG0387ER60548, awarded by the Department of Energy, and Grant No.NS35311, awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] This invention is directed to methods for detecting polymorphismsin complex eukaryotic genes, particularly the gene for ataxiatelangiectasia, and to polymorphisms detected by those methods.

[0004] Many autosomal recessive genetic disorders are caused bymutations in complex single genes that cause the genes to malfunction,producing a defective product or no product at all. Many of these genesinclude multiple exons, promoters, and other significant regions.

[0005] Ataxia-telangiectasia (A-T) (MIM208900) is an autosomal recessivedisorder characterized by progressive cerebellar degeneration,immunodeficiency, growth retardation, premature aging, chromosomalinstability, acute sensitivity to ionizing radiation, and cancerpredisposition (R. A. Gatti, “Ataxia-Telangiectasia” in Genetic Basis ofHuman Cancer (Vogelstein & Kinzler, eds. McGraw-Hill, New York, 1998)).

[0006] The gene responsible for A-T, ATM, was initially localized tochromosome 11q23.1 (E. Lange et al., “Location of anAtaxia-Telangiectasia to a ˜500 kb Interval on Chromosome11q23.1:Linkage Analysis of 176 Families in an InternationalConsortium,” Am. J. Hum. Genet. 57:112-119 (1995); N. Uhrhammer et al.,“Sublocalization of an Ataxia-Telangiectasia Gene Distal to D11 S384 byAncestral Haplotyping in Costa Rican Families,” Am. J. Hum. Genet.57:103-111 (1995)) and, on this basis, was positionally cloned bySavitsky et al. (K. Savitsky et al., “A Single Ataxia-TelangiectasiaGene with a Product Similar to a PI-3 Kinase,” Science 268:1749-1753(1995)). It spans about 150 kb of genomic DNA, encodes a majortranscript of 13 kb, and a 370 kDa protein (G. Chen & E. Y. H. P. Lee,“The Product of the ATM Gene is a 370-kDa Nuclear Phosphoprotein,” J.Biol. Chem. 271:33693-33697 (1996)). Subsequently, a wide spectrum ofATM mutations has been detected in A-T patients, spread throughout thegene and without evidence of a mutational hot spot (P. Concannon & R. A.Gatti, “Diversity of ATM Gene Mutations in Patients withAtaxia-Telangiectasia,” Hum. Mutat. 10:100-107 (1997)).

[0007] Procedures used for mutation screening in the ATM gene haveincluded restriction-endonuclease fingerprinting (REF) (K. Savitsky etal. supra (1995); P. J. Byrd et al., “Mutations Revealed by Sequencingthe 5′ Half of the Gene for Ataxia-Telangiectasia,” Hum. Mol. Genet.5:145-149 (1996)), the single-strand conformation polymorphism (SSCPTechnique) J. Wright et al., “A High Frequency of Distinct ATM Mutationsin Ataxia in Telangiectasia,” Am. J. Hum. Genet. 59:839-846 (1996); T.Sasaki et al., “ATM Mutations in Patients with Ataxia-TelangiectasiaScreened by a Hierarchical Strategy,” Hum. Mutat. 12:186-195 (1998)),and the protein truncation test (PTT); (M. Telatar et al.,“Ataxia-Telangiectasia: Mutations in ATM cDNA Detected byProtein-Truncation Screening,” Am. J. Hum. Genet. 59:40-44 (1996)).

[0008] The ATM gene shows homology with protein kinases in yeast(TEL-1), drosophila (Mei-41) and human (DNA-PK) and is most closelyrelated to DNA-PK and TEL-1(Savitsky et al., (1995), supra; K. Savitskyet al., Hum. Mol. Genet. 4:2025-2032 (1995); Lehmann et al., TrendsGenet. 11:375-377 (1995); Zakin, Cell 82:685-687 (1995); Lavin et al.,Trends Biol. Sci. 20:382-383 (1995); Keith et al., Science 270:50-51(1995)).

[0009] The nucleotide sequence encoding the ATM protein is SEQ ID NO: 1.This corresponds to GenBank Accession No. U33841. The open reading frameis 9168 nucleotides. There is a 3′ untranslated region (UTR) and a 5′UTR. SEQ ID NO: 2 is the amino acid sequence of the deduced ATM protein.It has 3056 amino acids. The ATM gene product contains aphosphatidylinositol-3 kinase (PI-3) signature sequence at codons2855-2875. Mutation analyses in the initial report by Savitsky et al.(K. Savitsky et al. (1995), supra) use restriction endonucleasefingerprinting to identify mutations in the reverse-transcribed 5.9 kbcarboxy-terminal end, which included the PI-3 signature sequence, of the10 kb transcript that was available at that time (K. Savitsky et al.,Hum. Mol. Genet. 4:2025-2032 (1995)). Both in-frame and frameshiftmutations were found. Because the methodology used for screening formutations biases the types of mutations found, there is a need to usedifferent screening methods to identify further mutations in the ATMgene. The complete 150 kb genomic sequence was subsequently published(M. Platzer et al., “Ataxia-Telangiectasia Locus: Sequence Analysis of184 kb of Human Genomic DNA Containing the entire ATM Gene,” Genome Res.7: 592-605 91988) and assigned Accession Number V82828.

[0010] The ATM gene is an example of a complex polyexonic eukaryoticgene that codes for a large protein product, in which defects appear asautosomal recessive mutations. There exists a large number of clinicallyimportant genes of this category, and improved methods of detectingpolymorphisms in such genes are needed. In particular, there is a needfor methods that can use either DNA or RNA as starting materials so thatthey are not dependent on existence of RNA molecules. Previoustechniques include restriction endonuclease fingerprinting (REF), thesingle-stranded conformation polymorphism (SSCP) technique and theprotein truncation test (PTT). There is also a need for a method thatcan detect mutations occurring in non-coding regions such as controlelements, which would be missed by the protein truncation test.Therefore, there is a need for improved methods of detection ofmutations and polymorphisms in such complex polyexonic eukaryoticstructural genes.

[0011] Because of the severity of the disease associated with mutationsin the ATM gene, patients or families frequently request confirmation ofa suspected diagnosis of A-T. If the mutation is already known in afamily, it is much easier to test other family members to see whetherthey carry that mutation. Since carriers of ATM mutations (i.e.,heterozygotes with one normal gene) may also be at an increased risk ofcancer, particularly breast cancer, testing for such mutations hasattracted much commercial interest. Automated chips and readers arebeing developed by many companies; however, these readers have an errorrate of about 1/1000, making it difficult to distinguish real mutationsfrom errors or normal variations (i.e., polymorphisms). Approximately23,000 nucleotides must be screened to identify most ATM mutations. Anormal polymorphism appears every 500 nucleotides. Thus, in a region of23,000 nucleotides being searched, there should be one (or possibly two)mutations amidst 23+46+2=71 errors and polymorphisms. The interpretationof such information is best approached by “look-up” tables that list allknown polymorphisms and mutations (sometimes referred to as SNPs orsingle nucleotide polymorphisms. Therefore, there is a need for improvedmethods of detecting polymorphisms in the ATM gene and in other large,complex, polyexonic genes in order to improve such automated screening.

SUMMARY

[0012] One aspect of the present invention is method of detecting amutation or a polymorphism in the human ataxia telangiectasia genecomprising the steps of:

[0013] (1) amplifying a plurality of nonoverlapping nucleic acidsegments from the human ataxia telangiectasia gene;

[0014] (2) subjecting the amplified nonoverlapping nucleic acid segmentsto single-stranded conformation polymorphism electrophoresis in a numberof lanes such that two or three amplified nucleic acid segments areelectrophoresed per lane, the electrophoresis of the segmentselectrophoresed in the same lane being initiated at different times,such that the signals from each amplified nucleic acid segment aredistinct in each lane, the time interval between the initiation ofelectrophoresis for each segment being chosen to ensure that signalsresulting from the electrophoresis are distinct for each segmentelectrophoresed in the same lane; and

[0015] (3) comparing the signals from the resulting single-strandedconformation polymorphism electrophoresis for each segment in each ofthe lanes to detect the mutation or polymorphism.

[0016] The plurality of nonoverlapping nucleic acid segments that areamplified can be RNA and the segments can be amplified by the reversetranscriptase-polymerase chain reaction mechanism. Alternatively, theplurality of nonoverlapping nucleic acid segments that amplified can beDNA and the segments can be amplified by the polymerase chain reactionmechanism.

[0017] The method preferably further comprises the step of cleavingamplified products larger than about 350 bases with a restrictionendonuclease that cleaves the amplified products into fragments that areless than about 350 bases.

[0018] Typically, the electrophoresis occurs in polyacrylamide gels withglycerol as a gel matrix from about 150 to about 250 volts for about 14to 16 hours. Preferably, the electrophoresis is performed in a pluralityof gels so that the step of comparing the signals resulting from theelectrophoresis of the amplified nucleic acid segments can detectmutations or polymorphisms in a plurality of segments of the gene. Thisprocedure is entitled mega-SSCP.

[0019] A set of 70 primers can be used, as shown in Table 1.

[0020] The method can also be applied to the detection of mutations orpolymorphisms in other genes. These genes include the APC gene, the CFTRgene, the BRCA1 gene, the BRCA2 gene, the HBB gene, the APOE gene, thePRNP gene, the SCA1 gene, the APP gene, the HPRT gene, the PAX3 gene,the RET gene, the PMP22 gene, the SCN4A gene, and the GNAS1 gene.

[0021] Another aspect of the present invention is an isolated andpurified nucleic acid fragment comprising nucleic acid havingcomplementarity or identity to a mutation in the ataxia-telangiectasiamutated (ATM) gene, the mutation being selected from the groupconsisting of:

[0022] (1) 10744A>G;

[0023] (2) 11482G>A;

[0024] (3) IVS3-558A>T;

[0025] (4) 146C>G;

[0026] (5) 381delA;

[0027] (6) IVS8-3delGT

[0028] (7) 1028delAAAA

[0029] (8) 1120C>T;

[0030] (9) 1930ins16

[0031] (10) IVS16+2T>C;

[0032] (11) 2572T>C;

[0033] (12) IVS21+1G>A;

[0034] (13) 3085delA;

[0035] (14) 3381delTGAC;

[0036] (15) 3602delTT;

[0037] (16) 4052delT;

[0038] (17) 4396C>T;

[0039] (18) 5188C>T;

[0040] (19) 5290delC;

[0041] (20) 5546delT;

[0042] (21) 5791G>CCT;

[0043] (22) 6047A>G;

[0044] (23) IVS44-1G>T;

[0045] (24) 6672delGC/6677delTACG;

[0046] (25) 6736del11/6749del7;

[0047] (26) 7159insAGCC;

[0048] (27) 7671delGTTT;

[0049] (28) 7705del14

[0050] (29) 7865C>T;

[0051] (30) 7979delTGT;

[0052] (31) 8177C>T;

[0053] (32) 8545C>T;

[0054] (33) 8565T>A;

[0055] (34) IVS64+1G>T; and

[0056] (35) 9010del28.

[0057] Yet another aspect of the present invention is an isolated andpurified nucleic acid fragment comprising nucleic acid havingcomplementarity or identity to a polymorphism or SNP in theataxia-telangiectasia mutated (AIM) gene, the polymorphism beingselected from the group consisting of:

[0058] (1) 10807A>G;

[0059] (2) IVS3-122T>C;

[0060] (3) IVS6+70delT;

[0061] (4) IVS16-34C>A;

[0062] (5) IVS22-77T>C;

[0063] (6) IVS24-9delT;

[0064] (7) IVS25-13delA;

[0065] (8) 5557G>A;

[0066] (9) IVS48-69insATT; and

[0067] (10) IVS62-55T>C.

[0068] These polymorphisms are relatively common polymorphisms.

[0069] Yet another aspect of the present invention is an isolated andpurified nucleic acid fragment comprising nucleic acid havingcomplementarity or identity to a polymorphism in theataxia-telangiectasia mutated (ATM) gene, the polymorphism beingselected from the group consisting of:

[0070] (1) 10677G>C;

[0071] (2) 10742G>T;

[0072] (3) 10819G>T;

[0073] (4) 10948A>G;

[0074] (5) IVS3-300G>A;

[0075] (6) IVS8-24del5;

[0076] (7) IVS13-137T>C;

[0077] (8) IVS14-55T>G;

[0078] (9) 1986T>C;

[0079] (10) IVS20+27delT;

[0080] (11) IVS23-76T>C;

[0081] (12) IVS25-35T>A;

[0082] (13) IVS27-65T>C;

[0083] (14) IVS30-54T>C;

[0084] (15) 4362A>C;

[0085] (16) IVS38-8T>C;

[0086] (17) 5793T>C;

[0087] (18) IVS47-11G>T;

[0088] (19) IVS49-16T>A;

[0089] (20) IVS53+34insA;

[0090] (21) IVS60-50 delTTAGTT;

[0091] (22) IVS62+8A>C;

[0092] (23) IVS62-65G>A; and

[0093] (24) 9200C>G.

[0094] These are relatively rare polymorphisms.

[0095] Another aspect of the present invention is a method for testing aDNA sample of a human for the presence or absence of a mutation orpolymorphism in the ATM gene comprising the steps of:

[0096] (1) providing a sample of DNA from a human; and

[0097] (2) testing the sample for the presence of a mutation or apolymorphism in the ATM gene, the mutation or the polymorphism being oneof the mutations or polymorphisms described above.

[0098] Yet another aspect of the present invention is an isolated andpurified protein, polypeptide, or peptide encoded by a polynucleotidethat comprises one of the fragments described above.

[0099] Still another aspect of the present invention is an antibody thatspecifically binds the isolated and purified protein, polypeptide, orpeptide.

[0100] Another aspect of the present invention is a transgenic mammalall of whose germ cells and somatic cells contain the fragment describedabove introduced into the mammal or an ancestor of the mammal at anembryonic stage. Typically, the transgenic mammal is a mouse.

BRIEF DESCRIPTION OF THE DRAWINGS

[0101] These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription, appended claims, and accompanying drawings where:

[0102]FIG. 1 is an electropherogram showing the results of sequentialloading of three sets of ATM PCR fragments, demonstrating no overlap ofSSCP patterns; from left to right: A), exons 34, 32, and 35; B), exons23, 22, and 20; C), exons 45, 60, and 62; by way of example, the loadingtimes for the three sets of PCR products shown in A) were t=0, exon 34;t=45 min, exon 32; t=75 min, exon 35; and

[0103]FIG. 2 is an electropherogram showing the results of mega-SSCP,one of three gels used to screen 70 PCR fragments from two patients bySSCP is shown; the arrow indicates an abnormal pattern, which isselected to be sequenced.

DESCRIPTION Definitions

[0104] As used herein, the terms defined below have the followingmeanings unless otherwise indicated:

[0105] “Nucleic Acid Sequence”: the term “nucleic acid sequence”includes both DNA and RNA unless otherwise specified, and, unlessotherwise specified, includes both double-stranded and single-strandednucleic acids. Also included are hybrids such as DNA-RNA hybrids. Inparticular, a reference to DNA includes RNA that has either theequivalent base sequence except for the substitution of uracil in RNAfor thymine in DNA, or has a complementary base sequence except for thesubstitution of uracil for thymine, complementarity being determinedaccording to the Watson-Crick base pairing rules. Reference to nucleicacid sequences can also include modified bases as long as themodifications do not significantly interfere either with binding of aligand such as a protein by the nucleic acid or with Watson-Crick basepairing. Reference to nucleic acid sequences also can include nucleicacid sequences that are conjugated or linked covalently or noncovalentlyto other chemical moieties such as proteins, fluorescers, or otherlabels as long as the other chemical moieties do not significantlyinterfere either with binding of a ligand by the nucleic acid or withWatson-Crick base pairing as appropriate for the particular nucleic acidsequence. Reference to nucleic acid sequences also includes thecomplementary nucleic acid sequence according to the Watson-Crick basepairing rules unless otherwise specified.

[0106] “Antibody”: as used herein the term “antibody” includes bothintact antibody molecules of the appropriate specificity, and antibodyfragments (including Fab, F(ab′), Fv, and F(ab′)₂), as well aschemically modified intact antibody molecules and antibody fragments,including hybrid antibodies assembled by in vitro-reassociation ofsubunits. Also included are single-chain antibody molecules generallydenoted by the term sFv and humanized antibodies in which some or all ofthe originally non-human constant regions are replaced with constantregions originally derived from human antibody sequences. Bothpolyclonal and monoclonal antibodies are included unless otherwisespecified. Additionally included are modified antibodies or antibodiesconjugated to labels or other molecules that do not block or alter thebinding capacity of the antibody.

Description

[0107] One aspect of the present invention is a method for the detectionof mutations and polymorphisms in complex, multiexonic eukaryotic genes,such as the ATM gene. Another aspect of the present invention isdirected to new mutations and polymorphisms detected in the ATM gene.

[0108] I. Methods for Detection of Mutations and Polymorphisms

[0109] An improved method for detection of polymorphisms and mutationsin large polyexonic eukaryotic genes that encode a large proteinmolecule, defects in which lead to the existence of autosomal recessivemutations, employs an improvement in the single-stranded conformationpolymorphism (SSCP) technique, known as mega-SSCP. This techniqueemploys the use of a gel electrophoresis technique that allows therunning of multiple samples in the same gel electrophoresis lane in thesingle-strand conformation polymorphism technique. Therefore, thisallows the screening of a large number of nucleic acid segments.

[0110] Although this technique is of general application, it isdescribed specifically with respect to detection of mutations orpolymorphisms in the ataxia-telangiectasia (ATM) gene.

[0111] As applied to the ATM gene, the method of detecting the mutationor polymorphism comprises the steps of:

[0112] (1) amplifying a plurality of nonoverlapping nucleic acidsegments from the human ataxia-telangiectasia gene;

[0113] (2) subjecting the amplified nonoverlapping nucleic acid segmentsto single-stranded conformation polymorphism electrophoresis in a numberof lanes such that two or three amplified nucleic acid segments areelectrophoresed per lane, the electrophoresis of the segmentselectrophoresed in the same lane being initiated at different times,such that the signals from each amplified nucleic acid segment aredistinct in each lane, the time interval between the initiation ofelectrophoresis for each segment being chosen to ensure that signalsresulting from the electrophoresis are distinct from each segmentelectrophoresed in the same lane; and

[0114] (3) comparing the signals from the resulting single-strandedpolymorphism electrophoresis for each segment in each of the lanes todetect the mutation or polymorphism.

[0115] The mutation or polymorphism is detected by observing adifference in the position of the bands. The gel pattern used enablesthe person performing the assay to reference the pattern back to thematrix of samples electrophoresed to give positive identification of thesample where a difference in mobility signals the existence of asingle-stranded conformation polymorphism.

[0116] The single-stranded conformation polymorphism technique detectschanges in the conformation of single-stranded nucleic acidselectrophoresed under nondenaturing conditions. In such conditions, eachnucleic acid segment forms a distinct structure determined by theability of bases to pair with distant bases in the same strand. Mobilityis also influenced by the size of the molecule.

[0117] Thus, single-stranded conformation polymorphism can detectrelatively small differences in structure which are reflected in themobility of the nucleic acid segments being electrophoresed.

[0118] The nucleic acid segments that are amplified can be amplified bystandard techniques. The choice of techniques depends on the materialsavailable. If the starting material involved is DNA, the segments aretypically amplified by the polymerase chain reaction mechanism (PCR) asdescribed in U.S. Pat. No. 4,683,195 to Mullis and U.S. Pat. No.4,684,202 to Mullis, incorporated herein by this reference.

[0119] This is the method of choice when genomic DNA is available.Conditions for polymerase chain reaction amplification are generallyknown in the art and need not be described further here. They aredescribed, for example, in M. I. Innis et al., eds., “PCR Protocols: AGuide to Methods and Applications” (Academic Press, San Diego, 1990),incorporated herein by this reference.

[0120] Other amplification techniques such as the ligase amplificationreaction (LAR) can alternatively be used.

[0121] If the starting material available is RNA, typically messengerRNA, the preferred technique is reverse transcriptase-polymerase chainreaction amplification. Basically, this method involves transcribing DNAfrom RNA using a retroviral reverse transcriptase, and then using theDNA transcribed for amplification according to the polymerase-chainreaction mechanism.

[0122] Conditions for electrophoresis can be chosen by one of ordinaryskill in the art according to the size of the nucleic acid segments tobe electrophoresed. Preferred conditions are described in M. Orita etal., “Detection of Polymorphisms of Human DNA by Gel Electrophoresis asSingle-Strand Conformation Polymorphisms,” Proc. Natl. Acad. Sci. USA86: 2766-2770 (1989). Preferably, electrophoresis is performed in apolyacrylamide gel of about 3% to about 7% polyacrylamide, morepreferably of about 4% to about 6% polyacrylamide, most preferably ofabout 5% polyacrylamide. Preferably, the gel is prepared in glycerol.Most preferably, the glycerol concentration is about 10% Preferably, thegels are run at about 4° C. using a system that maintains a constanttemperature and recirculates the buffer. A preferred buffer is 90 mMTris-borate, pH 8.3, 4 mM EDTA. Preferred running conditions are fromabout 150 to 250 volts for about 14-16 hours, depending on the size ofthe analyzed fragments. The polyacrylamide can be replaced with aproprietary gel matrix called “Mutation Detection Enhancement” from FMCBioproducts.

[0123] The nucleic acid segments that have been amplified andelectrophoresed can be detected by standard techniques that arewell-known to those skilled in the art. A preferred technique is silverstaining. A preferred method of silver staining involves rinsing the gelwith 10% ethanol for 10 minutes, with 1% nitric acid for three minutes,performing two quick rinses with distilled water for 30 seconds, silvernitrate for 20 minutes, two very quick rinses with distilled water,2.96% sodium carbonate/0.054% formaldehyde (37%) for developing, and 10%acetic acid for 10 minutes. Other staining techniques can alternativelybe used.

[0124] If the amplified fragments are larger than about 350 base pairs,they are typically cleaved with a restriction endonuclease that cleavesthe amplified products into fragments that are less than about 350bases. The restriction endonuclease can be chosen depending on thesequence to be cleaved and the frequency of cleavage required. Suitablerestriction endonucleases for use in cleaving amplified fragments areknown in the art and are described, for example, in J. Sambrook et al.,“Molecular Cloning: A Laboratory Manual” (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), vol. 1, pp. 5.3-5.9,incorporated herein by this reference. Such restriction endonucleasesrecognize sequences of four or more bases appearing in the amplifiedfragments and cleave at a defined point. Generally, the defined point iswithin the recognized sequence, but, in some cases, the defined pointcan be outside of the recognized sequence. Many enzymes recognizepalindromic sequences of from 4 to 8 bases. The cleavage points and thelengths of the resulting products can be predicted from the sequence ofthe nucleic acid and from the known specificity of the enzyme to be usedfor cleavage.

[0125] Typically, the electrophoresis is performed in a plurality ofgels so that the step of comparing the signals resulting from theelectrophoresis of the amplified nucleic acid segments can detectmutations or polymorphisms in a plurality of segments of the gene.

[0126] Typically, for detection of mutations or polymorphisms in the ATMgene on two persons, three gels are used and a total of 70 segments areamplified and electrophoresed using the single-stranded conformationpolymorphism technique.

[0127] For the ATM gene the primers that are preferred to be used are asshown in Table 1, below: TABLE 1 Fragment Forward Reverse Promoter 1(SEQ ID NO: 3) (SEQ ID NO: 4) 5′TCCGCGCTTACCC 5′ATGGCCAGCGACT AATA-3′TAGC-3′ Promoter 2 (SEQ ID NO: 5) (SEQ ID NO: 6) 5′AAGAGGGTGGGTG5′CACTCGGAAGGTC AGAG-3′ AAAG-3′ Exon 1a (SEQ ID NO: 7) (SEQ ID NO: 8)5′CGACGGGCCGAAT 5′AGGAGAGGGAGG GTTTGG-3′ AGTCAAGG-3′ Exon 1b (SEQ ID NO:9) (SEQ ID NO: 10) 5′CCTCTCCTCACTCC 5′CTTCCGTTATGAC ATCT-3′ TGTTTCG-3′Exon 2 (SEQ ID NO: 11) (SEQ ID NO: 12) 5′CGAAACAGTCATA 5′GATAAAAGGAAAACGGAAG-3′ AACAATACTA-3′ Exon 3 (SEQ ID NO: 13) (SEQ ID NO: 14)5′GTTTATCTAAAAT 5′GATGCAAACAATA GATTCTCTC-3′ TTTACTACT-3′ Intron 3a (SEQID NO: 15) (SEQ ID NO: 16) 5′CTCTGATAACCTC 5′GAATAGAAAACAGCTACTT-3′CCAGGTA-3′ Intron 3b (SEQ ID NO: 17) (SEQ ID NO: 18)5′AATGTTAAATCCT 5′CACAAAAATGTTT TGAGTGCT-3′ GCCTTGCT-3′ Exon 4 (SEQ IDNO: 19) (SEQ ID NO: 20) 5′TTAATCCTGCTAC 5′TGAAAATAAAAAG TACTGC-3′GAAATAATGG-3′ Exon 5 (SEQ ID NO: 21) (SEQ ID NO: 22) 5′CAGAACGAAAGGT5′ATATATAGGAAGC AGTAAATT-3′ AAAGATAAATG-3′ Exon 6 (SEQ ID NO: 23) (SEQID NO: 24) 5′GTAATCTAAGCAA 5′GTACTTACACTCA GGTGGT-3′ ACTTTTATCTT-3′ Exon7 (SEQ ID NO: 25) (SEQ ID NO: 26) 5′GCCATTCCAAGTG 5′TCACAAACAACAATCTTA-3′ CCTTCA-3′ Exon 8 (SEQ ID NO: 27) (SEQ ID NO: 28)5′AAATCCT~TTTCTG 5′TACTGAGTCTAAA TATGGG-3′ ACATGGTCT-3′ Exon 9 (SEQ IDNO: 29) (SEQ ID NO: 30) 5′AGTGTGAAGTAAT 5′TCAACCAGAGAAA GCTGTGAT-3′TCCAGAG-3′ Exon 10 (SEQ ID NO: 31) (SEQ ID NO: 32) 5′CCAGGTGTCTTCT5′TTATAGGCTTTTTG AACG-3′ TGAGAAC-3′ Exon 11 (SEQ ID NO: 33) (SEQ ID NO:34) 5′GGTTGTGGTGATA 5′CTGGTTGAGATGA CGAG-3′ AAGGAT-3′ Exon 12 (SEQ IDNO: 35) (SEQ ID NO: 36) 5′GTACTATGGAAAT 5′CAGGGATATGTGA GATGGTG-3′GTGTG-3′ Exon 13 (SEQ ID NO: 37) (SEQ ID NO: 38) 5′GGCACTGTCCTGA5′GCATCAAATAAGT TAGAT-3′ GGAGA-3′ Exon 14 (SEQ ID NO: 39) (SEQ ID NO:40) 5′CAATGGTTGTCCT 5′AGATGCAGCTACT CCTTAA-3′ ACCC-3′ Exon 15 (SEQ IDNO: 41) (SEQ ID NO: 42) 5′GTCCGAAGAAGAG 5′CTATTTCTCCTTCC AAGC-3′TAACAG-3′ Exon 16 (SEQ ID NO: 43) (SEQ ID NO: 44) 5′GTTCTTACAAAAG5′GTCTTCCAAACAA ATAGAGT-3′ ATGTAAT-3′ Exon 17 (SEQ ID NO: 45) (SEQ IDNO: 46) 5′GTACACTGTAAAA 5′GAGGTCAAGGCTA AGCAATAC-3′ CAATG-3′ Exon 18(SEQ ID NO: 47) (SEQ ID NO: 48) 5′ACATTCCATTCAA 5′GCTATATGTTGTGGATAGAGA-3′ AGATGC-3′ Exon 19 (SEQ ID NO: 48) (SEQ ID NO: 50)5′AAATTTTGACTAC 5′CCTCTTATACTGC AGCATGCT-3′ CAAATC-3′ Exon 20 (SEQ IDNO: 51) (SEQ ID NO: 52) 5′ACTATAATTTTGC 5′CATTTAGTCAGCA TTTTCATATACT-3′ACATCAG-3′ Exon 21 (SEQ ID NO: 53) (SEQ ID NO: 54) 5′TTAAAGTAAATGA5′CTTAACAGAACAC TTTGTGGAT-3′ ATCAGT-3′ Exon 22 (SEQ ID NO: 55) (SEQ IDNO: 56) 5′CTGAAACCACTAT 5′TTGCATTCGTATC CGTAAGA-3′ CACAGA-3′ Exon 23(SEQ ID NO: 57) (SEQ ID NO: 58) 5′AAAGACATATTGG 5′AGCCTACGGGAAAAAGTAACTTA-3′ AGAACT-3′ Exon 24 (SEQ ID NO: 59) (SEQ ID NO: 60)5′AGTAAGATCTCCA 5′CATTCTACTGCCA TTGAAAATTT-3′ TCTGC-3′ Exon 25 (SEQ IDNO: 61) (SEQ ID NO: 62) 5′GTGATTTATTTTGT 5′CATACAGTTGTTT TCTGGAATA-3′TAGAGCAG-3′ Exon 26 (SEQ ID NO: 63) (SEQ ID NO: 64) 5′TGGAGTTCAGTTTG5′GTGCCACTCAGAA GGATTT-3′ AATCTA-3′ Exon 27 (SEQ ID NO: 65) (SEQ ID NO:66) 5′AAGAAAAGTTGAA 5′TGTGTATGGGTAT TGAATGTTGTT-3′ GGTATG-3′ Exon 28(SEQ ID NO: 67) (SEQ ID NO: 68) 5′GATACTTTAATGC 5′CGAATAAATCGAATGATGGTA-3′ TAAATAGCC-3′ Exon 29 (SEQ ID NO: 69) (SEQ ID NO: 70)5′GTCATCGAATACT 5′CTCAATTCAAAGG TTTGGAAA-3′ TGGCTAT-3′ Exon 30 (SEQ IDNO: 71) (SEQ ID NO: 72) 5′CATTTTGGAAGTT 5′CCTCTTTAAGATG CACTGG-3′TATTTACAA-3′ Exon 31 (SEQ ID NO: 73) (SEQ ID NO: 74) 5′ATATCAAACCCAA5′AAAAAACAGGAA ATCTAAATTCT-3′ GAACAGGAT-3′ Exon 32 (SEQ ID NO: 75) (SEQID NO: 76) 5′AGATGCTGAACAA 5′AACACTCAAATCC AAGGACT-3′ TTCTAACA-3′ Exon33 (SEQ ID NO: 77) (SEQ ID NO: 78) 5′GTTTTGTTGGCTTA 5′GAGCATTACAGATCTTT-3′ TTTTG-3′ Exon 34 (SEQ ID NO: 79) (SEQ ID NO: 80) 5′GTCTATAAATGGC5′TGACAATGAAACC ACTTAACT-3′ AAGAGC-3′ Exon 35 (SEQ ID NO: 81) (SEQ IDNO: 82) 5′CAATTATAAACAA 5′ACTACAGGCAACA AAGTGTTGTCT-3′ GAAAACA-3′ Exon36 (SEQ ID NO: 83) (SEQ ID NO: 84) 5′TGAAGTACAGAAA 5′GTGTGAAGTATCAAACAGCAT-3′ TTCTCCAT-3′ Exon 37 (SEQ ID NO: 85) (SEQ ID NO: 86)5′GGTGTACTTGATA 5′TGTTTTAGATATG GGCATTT-3′ CTGGG-3′ Exon 38 (SEQ ID NO:87) (SEQ ID NO: 88) 5′TACAATGATTTCC 5′TATTATGTGAAGA ACTTCTCT-3′TGATGTGC-3′ Exon 39 (SEQ ID NO: 89) (SEQ ID NO: 90) 5′TCATTTTTACTCAA5′CCATCTTAAATCC ACTATTG-3′ ATCTTTCT-3′ Exon 40 (SEQ ID NO: 91) (SEQ IDNO: 92) 5′TTATAGCATAGTG 5′TTTGCAACACCTT GGAGACA-3′ CACCTAA-3′ Exon 41(SEQ ID NO: 93) (SEQ ID NO: 94) 5′TAAGCAGTCACTA 5′TATACCCTTATTGCCATTGTA-3′ AGACAATGC-3′ Exon 42 (SEQ ID NO: 95) (SEQ ID NO: 96)5′GTATTCAGGAGCT 5′ATGGCATCTGTAC TC-3′ AGTGTCT-3′ Exon 43 (SEQ ID NO: 97)(SEQ ID NO: 98) 5′TTGTTGTTTCCATG 5′TGCTTCGTGTTCAT TTTTCAGG-3′ ATGTTCG-3′Exon 44 (SEQ ID NO: 99) (SEQ ID NO: 100) 5′GTGGTGGAGGGAA 5′CTGAAATAACCTCGATGTTA-3′ AGCACTACA-3′ Exon 45 (SEQ ID NO: 101) (SEQ ID NO: 102)5′TGTATCTTTGCTGT 5′CAGTTGTTGTTTA TTTTTTC-3′ GAATGAG-3′ Exon 46 (SEQ IDNO: 103) (SEQ ID NO: 104) 5′CATGTATATCTTA 5′CTTCATCAATGCA GGGTTCTG-3′AATCCTTACA-3′ Exon 47 (SEQ ID NO: 105) (SEQ ID NO: 106) 5′CAAAGCCTATGAT5′CCCACTTCAGCCT GAGAAC-3′ TCTAAA-3′ Exon 48 (SEQ ID NO: 107) (SEQ ID NO:108) 5′TTTTTCATTTCTCT 5′GACATTTCTTTTTC TGCTTACAT-3′ CCTCAG-3′ Exon 49(SEQ ID NO: 109) (SEQ ID NO: 110) 5′GGTAGTTGCTGCT 5′AAATTACTAATTTTTCATT-3′ CAAGGCTCTA-3′ Exon 50 (SEQ ID NO: 111) (SEQ ID NO: 112)5′ACATTTTTAACCT 5′CCATACTTTTCTTT GCTTTTTTCC-3′ GCTTTGGAA-3′ Exon 51 (SEQID NO: 113) (SEQ ID NO: 114) 5′CCTTAATTTGAGT 5′ATGCAAAAACACTGATTCTTTAG-3′ CACTCAG-3′ Exon 52 (SEQ ID NO: 115) (SEQ ID NO: 116)5′AGTTCATGGCTTT 5′GTATACACGATTC TGTGTTTT-3′ CTGACAT-3′ Exon 53 (SEQ IDNO: 117) (SEQ ID NO: 118) 5′TAGTTAGTGAAGT 5′TTTGTATTTCCATT TTTGTTAAC-3′TCTTAG-3′ Exon 54 (SEQ ID NO: 119) (SEQ ID NO: 120) 5′AAGCAAAATGAAA5′GGAAAGACTGAAT AATATGG-3′ ATCACAC-3′ Exon 55 (SEQ ID NO: 121) (SEQ IDNO: 122) 5′GAAGTTTAAATGT 5′AGCAGATTTACTT TGGGTAG-3′ ATTAGGC-3′ Exon 56(SEQ ID NO: 123) (SEQ ID NO: 124) 5′GTGGTATCTGCTG 5′ACCAATTTTGACCACTATTC-3′ TACATAA-3′ Exon 57 (SEQ ID NO: 125) (SEQ ID NO: 126)5′GTTCTTAACCACT 5′CATTTCTACTCTAC ATCACATCGTC-3′ AAATCTTCCTCAT- Exon 58(SEQ ID NO: 127) (SEQ ID NO: 128) 5′ITGGTTTGAGTGC 5′TTCACCCAACCAACCTTTGC-3′ ATGGCAT-3′ Exon 59 (SEQ ID NO: 129) (SEQ ID NO: 130)5′TCAAATGCTCTTT 5′CAGCTGTCAGCTT AATGG-3′ TAATAAGCC-3′ Exon 60 (SEQ IDNO: 131) (SEQ ID NO: 132) 5′TCCTGTTCATCTTT 5′GCCAAACAACAAA ATTGCCCC-3′GTGCTCAA-3′ Exon 61 (SEQ ID NO: 133) (SEQ ID NO: 134) 5′GTGATTTCAGATT5′ATGATGACCAAAT GTTTGT-3′ ATTTACT-3′ Exon 62 (SEQ ID NO: 135) (SEQ IDNO: 136) 5′TGTGGTTTCTTGCC 5′CCAGCCCATGTAA TTTGT-3′ TTTTGA-3′ Exon 63(SEQ ID NO: 137) (SEQ ID NO: 138) 5′CTCTGCCAAGTAT 5′GACTTCCTGACGATATGCTATTT-3′ GATACACA-3′ Exon 64 (SEQ ID NO: 139) (SEQ ID NO: 140)5′TGTTTCTAAGTAT 5′CACTAAGGACAAA GTGATT-3′ AACACAAAGGT-3′ Exon 65 (SEQ IDNO: 141) (SEQ ID NO: 142) 5′TTAAACTGTTCAC 5′GGCAGGTTAAAAA CTCACT-3′TAAAGG-3′

[0128] The primers that amplify the exons also provide amplification ofthe adjoining intervening sequences (introns).

[0129] Other segments can be amplified with other primers.

[0130] In mega-SSCP, two or three amplified segments are electrophoresedin the same lane. The segments to be electrophoresed in the same laneare chosen empirically so that their mobilities are sufficientlydifferent so that the signals can be distinguished. These mobilities canbe determined by electrophoresing each of the amplified segmentsindividually to determine their mobilities in SSCP.

[0131] The methods that are described above with particular reference tothe ATM gene, a mutation in which causes the autosomal recessivedisorder ataxia-telangiectasia, can be extended to other genes, bothhuman and non-human. In general, such techniques can be applied todetection of a polymorphism in a polyexonic eukaryotic gene of at least4 kilobase pairs in length. The exact pattern of electrophoresis and theintervals between the start times for each sample are chosen from thesequence of the gene. The segments to be amplified, and in whichmutations or polymorphisms can therefore be detected, can include exons,introns, and promoter regions. This technique is of use in detectinggenes in which a mutation results in the presence of an autosomalrecessive condition, although it is not limited to such genes, and canbe used to detect autosomal dominant mutations as well.

[0132] An example of the sequential loading of the amplified segments isshown in FIG. 1. In FIG. 1, in A), amplification products of exons 34,32, and 35 are loaded; in B), amplification products of exons 23,-22,and 20 are loaded; and in C), amplification products of exons 45, 60,and 62 are loaded. By way of example, the loading times for the threesets of PCR products shown in FIG. 1A were: t=0, exon 34; t=45 min, exon32; t=75 min, exon 35.

[0133] Therefore, generally, the interval over which the segments areloaded ranges from 0 to 75 minutes.

[0134] The genes for which such methods are useful include, but are notlimited to, the CFTR gene, the APC gene, the BRCA1 gene, the BRCA2 gene,the HBB gene, the APOE gene, the PRNP gene, the SCA1 gene, the APP gene,the HPRT gene, the PAX3 gene, the RET gene, the PMP22 gene, the SCN4Agene, and the GNAS1 gene.

[0135] The CFTR gene is a gene in which a defect causes the autosomalrecessive disorder cystic fibrosis. The APC gene is a gene in whichmutations can cause the autosomal dominant condition familialadenomatous polyposis, in which colonic polyps develop which are likelyto become malignant. The BRCA1 and BRCA2 genes are genes in whichmutations predispose women to breast cancer. The HBB gene is a gene inwhich a defect causes the autosomal recessive disorder β-thalassemia.The APOE gene is a gene in which a defect can cause a number ofdisorders of lipid metabolism, leading, for example, tohypercholesterolemia and atherosclerosis. The PRNP gene is a gene inwhich a mutation causes Gerstmann-Sträussler-Scheinker syndrome. TheHPRT gene is a gene in which mutations can cause gout and/or Lesch-Nyhansyndrome, depending on the severity of the loss of function. The PAX3gene is a gene in which a loss-of-function mutation causes type 1Waardenberg syndrome. The RET gene is a gene in which a loss-of-functionmutation causes Hirschsprung disease. The PMP22 gene is a gene in whicha loss-of-function mutation causes Charcot-Marie-Tooth neuropathy type1A. The SCN4A gene is a gene in which mutations may cause severaldiseases, including paramyotonia congenita and hyperkalemic periodicparalysis. The GNAS1 gene is a gene in which mutations also may causeseveral conditions, including Albright hereditary osteodystrophy andMcCune-Albright syndrome. This list is illustrative and is not limiting.

[0136] These genes are examples of the genes in which mutations orpolymorphisms can be detected by the method of the present invention.

[0137] II. Isolated Nucleic Acid Fragments Encoding Mutations orPolymorphisms in the ATM Gene

[0138] Another aspect of the present invention is an isolated andpurified fragment comprising nucleic acid having complementarity oridentity to a mutation or to a polymorphism in the ATM gene. Themutation is one of those that is identified by the method describedabove in Section (I) and is one of those shown in Table 2. Thepolymorphism is one of those that is also identified by the methoddescribed above in Section (I) and is one of those shown in Table 3(relatively common polymorphisms) or Table 4 (rare polymorphisms). Thepolymorphisms in Table 3 are relatively common polymorphisms that occurat a frequency of from 14% to 45%. The polymorphisms in Table 4 arerelatively rare polymorphisms that occur at a frequency of from 0.5% to6%.

[0139] A fragment according to the present invention that isparticularly useful is a fragment that has complementarity to themutation or to the polymorphism in the ATM gene, is hairpin shaped, iscovalently linked to a fluorophore and to a quencher, and has astructure such that the fluorophore is internally quenched by thequencher when the fragment is not base-paired and such that the internalquenching is relieved when the fragment is base-paired, therebyrestoring fluorescence of the fluorophore. Such fragments are useful asmolecular beacons in detecting the mutations or polymorphisms (S. Tyagiet al., “Multicolor Molecular Beacons for Allele Discrimination,” NatureBiotechnol. 16: 49-53 (1998)). A particularly suitable quencher isDABCYL (4-[4′-dimethylaminophenylazo]benzoic acid). A large number ofsuitable fluorophores are known in the art, such as coumarin, EDANS.fluorescein, Lucifer yellow, BODIPY, tetramethylrhodamine, and Texasred. Further details on the methods are described in S. Tyagi et al.(1998), supra, incorporated herein by this reference.

[0140] Another fragment according to the present invention that isparticularly useful is a fragment that is DNA, that has complementarityto the mutation or to the polymorphism in the ATM gene and that hascovalently linked to either its 5′-end or to its 3′-end a segment ofabout 40 bases, the segment of about 40 bases comprising a repeatingunit of dCdG or dGdC. This fragment is useful as a primer that generatesa GC clamp on amplification by a procedure such as PCR. The resultingamplification product or amplicon has a sequence of dGdC in one strandand a sequence of dCdG in the other strand, thus generating a region ofexceptional stability to thermal denaturation by tethering one end. Thisincreases the overall melting range of the DNA molecule. This isparticularly useful for denaturing high performance liquidchromatography.

[0141] III. Methods of Testing DNA

[0142] Another aspect of the present invention is a method of testing aDNA sample for the presence of a mutation or polymorphism in the ATMgene. The mutation or polymorphism is one of those that is identified bythe method described above in Section (I) and is one of those shown inTable 2, Table 3 or Table 4.

[0143] In general, the method comprises:

[0144] (1) providing a sample of DNA from a human; and

[0145] (2) testing the sample for the presence or absence of themutation or polymorphism in the ATM gene.

[0146] Preferably, the step of testing the sample for the presence orabsence of the mutation comprises PCR amplifying one of the exons,introns, or control regions listed in Table I with the correspondingprimers and subjecting the PCR products to heteroduplex analysis todetect the presence or absence of the mutation. Methods for performingheteroduplex analysis are well known in the art and are described, forexample, in J. Keen et al., Trends Genet. 7:5 (1991). Heteroduplexanalysis is typically combined with SSCP.

[0147] Typically, the DNA sample comprises genomic DNA. It is alsopossible to test nongenomic DNA such as cDNA prepared by reversetranscription of RNA such as mRNA, but such DNA lacks exons and thus itis not possible to detect mutations in exons in such DNA samples.

[0148] In particular, the following mutations in the ATM gene aresubject to detection with the following combinations of primers:

[0149] (1) The mutation 10744A>G, with a primer set selected from thegroup consisting of either TCCGCGCTTACCCAATA (SEQ ID NO: 3) andATGGCCAGCGACTTAGC (SEQ ID NO: 4) or AAGAGGGTGGGTGAGAG (SEQ ID NO: 5) andCACTCGGAAGGTCAAAG (SEQ ID NO: 6).

[0150] (2) The mutation 11482G>A, with the primer set CCTCTCCTCACTCCATCT(SEQ ID NO: 9) and CTTCCGTTATGACTGTTTCG (SEQ ID NO: 10).

[0151] (3) The mutation IVS3-558A>T, with a primer set selected from thegroup consisting of either CTCTGATAACCTCCTACTT (SEQ ID NO: 15) andGAATAGAAAACAGCCAGGTA (SEQ ID NO: 16) or AATGTTAAATCCTTGAGTGCT (SEQ IDNO: 17) and CACAAAAATGTTTGCCTTGCT (SEQ ID NO: 18).

[0152] (4) The mutation 146C>G, with the primer setCAGAACGAAAGGTAGTAAATT (SEQ ID NO: 21) and ATATATAGGAAGCAAAGATAAATG (SEQID NO: 22).

[0153] (5) The mutation 381delA, with the primer set GCCATTCCAAGTGTCTTA(SEQ ID NO: 25) and TCACAAACAACAACCTT (SEQ ID NO: 26).

[0154] (6) The mutation IVS8-3delGT, with the primer setAAATCCTTTTTCTGTATGGG (SEQ ID NO: 27) and TACTGAGTCTAAAACATGGTCT (SEQ IDNO: 28).

[0155] (7) The mutation 1028delAAAA, with the primer setCCAGGTGTCTTCTAACG (SEQ ID NO: 31) and TTATAGGCTTTTTGTGAGAAC (SEQ ID NO:32).

[0156] (8) The mutation 1120C>T, with the primer set GGTTGTGGTGATACGAG(SEQ ID NO: 33) and CTGGTTGAGATGAAAGGAT (SEQ ID NO: 34).

[0157] (9) The mutation 1930ins16, with the primer set GTCCGAAGAAGAGAAGC(SEQ ID NO: 41) and CTATTTCTCCTTCCTAACAG (SEQ ID NO: 42).

[0158] (10) The mutation IVS16+2T>C, with the primer setGTTCTTACAAAAGATAGAGT (SEQ ID NO: 43) and GTCTTCCAAACAAATGTAAT (SEQ IDNO: 44).

[0159] (11) The mutation 2572T>C, with the primer setAAATTTTGACTACAGCATGCT (SEQ ID NO: 49) and CCTCTTATACTGCCAAATC (SEQ IDNO: 50).

[0160] (12) The mutation IVS21+1G>A, with the primer setTTAAAGTAAATGATTTGTGGAT (SEQ ID NO: 53) and CTTAACAGAACACATCAGT (SEQ IDNO: 54).

[0161] (13) The mutation 3085delA, with the primer setAAAGACATATTGGAAGTAACTTA (SEQ ID NO: 57) and AGCCTACGGGAAAAGAACT (SEQ IDNO: 58).

[0162] (14) The mutation 3381delTCAG, with the primer setGTGATTTATTTTGTTCTGGAATA (SEQ ID NO: 61) and CATACAGTTGTTTTAGAGCAG (SEQID NO: 62).

[0163] (15) The mutation 3602delTT, with the primer setAAGAAAAGTTGAATGAATGTTGTT (SEQ ID NO: 65) and TGTGTATGGGTATGGTATG (SEQ IDNO: 66).

[0164] (16) The mutation 4052delT, with the primer setGTCATCGAATACTTTTGGAAA (SEQ ID NO: 69) and CTCAATTCAAAGGTGGCTAT (SEQ IDNO: 70).

[0165] (17) The mutation 4396C>T, with the primer setATATCAAACCCAAATCTAAATTCT (SEQ ID NO: 73) and AAAAAACAGGAAGAACAGGAT (SEQID NO: 74).

[0166] (18) The mutation 5188C>T, with the primer setGGTGTACTTGATAGGCATTT (SEQ ID NO: 85) and TGTTTTAGATATGCTGGG (SEQ ID NO:86).

[0167] (19) The mutation 5290delC, with the primer setGGTGTACTTGATAGGCATTT (SEQ ID NO: 85) and TGTTTTAGATATGCTGGG (SEQ ID NO:86).

[0168] (20) The mutation 5549delT, with the primer setTCATTTTTACTCAAACTATTG (SEQ ID NO: 89) and CCATCTTAAATCCATCTTTCT (SEQ IDNO: 90).

[0169] (21) The mutation 5791G>CCT, with the primer setTAAGCAGTCACTACCATTGTA (SEQ ID NO: 93) and TATACCCTTATTGAGACAATGC (SEQ IDNO: 94).

[0170] (22) The mutation 6047A>G, with the primer setTTGTTGTTTCCATGTTTTCAGG (SEQ ID NO: 97) and TGCTTCGTGTTCATATGTTCG (SEQ IDNO: 98).

[0171] (23) The mutation IVS44-1G>T, with the primer setGTGGTGGAGGGAAGATGTTA (SEQ ID NO: 99) and CTGAAATAACCTCAGCACTACA (SEQ IDNO: 100).

[0172] (24) The mutation 6672delGG/6677delTACG, with the primer setTTTTTCATTTCTCTTGCTTACAT (SEQ ID NO: 107) and GACATTTCTTTTTCCCTCAG (SEQID NO: 108).

[0173] (25) The mutation 6736dell 1/6749del7, with the primer setTTTTTCATTTCTCTTGCTTACAT (SEQ ID NO: 107) and GACATTTCTTTTTCCCTCAG (SEQID NO: 108).

[0174] (26) The mutation 7159insAGCC, with the primer setCCTTAATTTGAGTGATTCTTTAG (SEQ ID NO: 113) and ATGCAAAAACACTCACTCAG (SEQID NO: 114).

[0175] (27) The mutation 7671delGTTT, with the primer setAAGCAAAATGAAAAATATGG (SEQ ID NO: 119) and GGAAAGACTGAATATCACAC (SEQ IDNO: 120).

[0176] (28) The mutation 7705del14, with the primer setAAGCAAAATGAAAAATATGG (SEQ ID NO: 119) and GGAAAGACTGAATATCACAC (SEQ IDNO: 120).

[0177] (29) The mutation 7865C>T, with the primer setGAAGTTTAAATGTTGGGTAG (SEQ ID NO: 121) and AGCAGATTTACTTATTAGGC (SEQ IDNO: 122).

[0178] (30) The mutation 7989delTGT, with the primer setGTGGTATCTGCTGACTATTC (SEQ ID NO: 123) and ACCAATTTTGACCTACATAA (SEQ IDNO: 124).

[0179] (31) The mutation 8177C>T, with the primer setTTGGTTTGAGTGCCCTTTGC (SEQ ID NO: 127) and TTCACCCAACCAAATGGCAT (SEQ IDNO: 128).

[0180] (32) The mutation 8545C>T, with the primer setTCCTGTTCATCTTTATTGCCCC (SEQ ID NO: 131) and GCCAAACAACAAAGTGCTCAA (SEQID NO: 132).

[0181] (33) The mutation 8565T>A, with the primer setTCCTGTTCATCTTTATTGCCCC (SEQ ID NO: 131) and GCCAAACAACAAAGTGCTCAA (SEQID NO: 132).

[0182] (34) The mutation IVS64+1G>T, with the primer setTGTTTCTAAGTATGTGATT (SEQ ID NO: 139) and CACTAAGGACAAAAACACAAAGGT (SEQID NO: 140).

[0183] (35) The mutation 9010del28, with the primer setTTAAACTGTTCACCTCACT (SEQ ID NO: 141) and GGCAGGTTAAAAATAAAGG (SEQ ID NO:142).

[0184] Other mutations and polymorphisms can be detected usingappropriate primer sets that cover the region of the mutation orpolymorphism for its amplification.

[0185] The mutations and polymorphisms in the ATM gene that are anaspect of the present invention are particularly useful forinterpretation in conjunction with automated high-densityoligonucleotide probes mounted on solid supports, sometimes referred toas “DNA chips,” as described in J. C. Hacia et al., “Strategies forMutational Analysis of the Large Multiexon ATM Gene using High-DensityOligonucleotide Arrays,” Genome Res. 8: 1245-1258 (1998).

[0186] IV. Isolated Protein, Polypeptide, or Peptide Products

[0187] Another aspect of the present invention is an isolated andpurified protein, polypeptide, or peptide encoded by a polynucleotidethat comprises one of the fragments listed above in Table 2, Table 3, orTable 4.

[0188] Such proteins, polypeptides, or peptides can be produced byincorporating the polynucleotide in a suitable vector operably linked toand under the control of one or more control sequences such aspromoters, operators, and enhancers, introducing the vector into asuitable compatible host in which the protein, polypeptide, or peptidecould be expressed, expressing the protein, polypeptide, or peptide, andisolating the protein, polypeptide, or peptide.

[0189] Such proteins, polypeptides, or peptides can also be produced bydetermining the sequence of the encoded protein, polypeptide, or peptideby the standard triplet genetic code and then directly synthesizing theprotein, polypeptide, or peptide, such as by standard solid-phasesynthesis methods.

[0190] V. Antibodies

[0191] Another aspect of the present invention is antibodies thatspecifically bind the proteins, polypeptides, or peptides of Section(IV). These antibodies can be prepared by standard methods. The proteinsand the larger polypeptides and peptides can be used directly asimmunogens, while, for the shorter peptides, it is generally preferredto couple them to a protein carrier such as keyhole limpet hemocyaninfor immunization.

[0192] The antibodies can be polyclonal or monoclonal. Monoclonalantibodies can be prepared by standard methods once antibody-producinganimals have been immunized with the proteins or polypeptides, or withthe peptides coupled to appropriate protein carriers.

[0193] VI. Transgenic Animals

[0194] Another aspect of the present invention is transgenic animals. Intransgenic animals according to the present invention, all of the germcells and the somatic cells of the animal contain one of the fragmentsof Section (II) introduced into the animal, or into an ancestor of theanimal, at an embryonic stage. Preferably, the transgenic animal is amouse, although other animals, such as rats, pigs, and sheep, can alsobe used.

[0195] Transgenic animals according to the present invention are usefulin determining the effects of the mutations in the fragments on thedevelopment of the organisms in which they are incorporated.

[0196] The invention is illustrated by the following Examples. TheseExamples are for illustrative purposes only and are not intended tolimit the invention.

EXAMPLES

[0197] Example 1

Detection of Mutations and Polymorphisms in the ATM Gene by Mega-SSCP

[0198] Patients and Methods

[0199] Patients

[0200] Ninety-two unrelated A-T patients from different populations(American, Turkish, Polish, Costa Rican, Canadian, and Spanish) werescreened. In most of the cases DNA was the only biological materialavailable from these patients. In a few samples, previous screening byPTT (M. Telatar et al., “Ataxia-Telangiectasia: Mutations in ATM cDNADetected by Protein-Truncation Screening,” Am J Hum Genet. 59:40-44(1996)) had failed to identify both mutations. In some experiments, DNAfrom 40 unrelated individuals (80 independent chromosomes) was used toscreen for and determine the allelic frequency of specificpolymorphisms.

[0201] Optimized Single-Strand Conformation Polymorphism (SSCP)Technique

[0202] An SSCP technique described previously by Orita et al. (M. Oritaet al. “Detection of Polymorphisms of Human DNA by Gel Electrophoresisas Single-Strand Conformation Polymorphisms,” Proc. Natl. Acad. Sci. USA86:2766-2770 (1989)) was optimized. Genomic DNA from 92 A-T patients anda normal individual was PCR-amplified for 70 different regions,including the 66 ATM exons (with additional 50-100 hp of flankingintronic sequence), the promoter region upstream of the first exon (P.J. Byrd et al., “Mutations Revealed by Sequencing the 5′ Half of theGene for Ataxiatelangiectasia,” Hum. Mol. Genet. 5:145-149 (1996)), anda putative promoter region within intron 3 (M. Platzer et al.,“Ataxia-Telangiectasia Locus: Sequence Analysis of 184 kb of HumanGenomic DNA Containing the Entire ATM Gene,” Genome Res. 7:592-605(1997)). If amplified products were larger than 350 hp they weredigested with a suitable restriction enzyme before being analyzed bySSCP.

[0203] All regions were analyzed by SSCP using MDE (Mutation DetectionEnhancement; FMC Bioproducts) with glycerol as the gel matrix. Gels wererun at 4° C. using the D-CODE Universal Mutation Detection System (BIORAD), which maintains a constant temperature and recirculates thebuffer. The running conditions varied between 150-250 V for 14-16 h,depending on the size of the analyzed fragments. Silver staining wasused to visualize the SSCP patterns (10% ethanol for 10 min, 1% HNO₃ for3 min, two quick rinses with distilled water for 30 sec, AgNO₃ for 20min, two very quick rinses with distilled water, 2.96% Na₂CO₃/0.054%formaldehyde 37% for developing, and 10% acetic acid for 10 min).

[0204] Due to the large number of DNA samples and PCR fragments,screening was expedited by sequentially loading 3 different sets of PCRproducts into the same gel. In order to do so, loading times werepretested and carefully established to keep SSCP patterns fromoverlapping (FIG. 1). The elapsed times between loading various PCRproducts ranged from 15 to 120 min. As controls, previously knownmutations were used that had been detected by the protein truncationtest (Telatar et al. (1996), supra) to optimize the efficiency of SSCPfor each PCR fragment.

[0205] DNA Sequencing

[0206] Once an abnormal pattern could be detected by SSCP, the specificfragment was PCR amplified, purified using a PCR purification column(Qiagen), and sequenced using the Thermo Sequenase Cy5.5 dye terminatorsequencing kit (Amersham) and the OpenGene DNA Automated SequencingSystem (Visible Genetics).

[0207] Primers

[0208] Primer sets used for each of the 70 PCR segments analyzed arelisted in Table 1, above.

[0209] Results

[0210] A TM Mutations

[0211] Before initiating SSCP screening of the new samples, the optimalconditions for detecting known mutations that had been previouslydefined in the amplified PCR fragments were determined (Telatar et al.(1996), supra; M. Telatar et al., “Ataxia-Telangiectasia: Identificationand Detection of Founder-Effect Mutations in the ATM gene in EthnicPopulations,” Am. J. Hum. Genet. 62:86-97 (1998) (“Telatar et al.(1998a)”). Abnormal SSCP patterns were observed for all known mutations.A total of 118 of 177 expected mutations were observed as present in thescreening (85 non-consanguineous patients with two unknown mutations and7 non-consanguineous patients with one unknown mutation). Therefore, theefficiency of mutation detection by optimized SSCP was ˜70%. Among thedetected mutations, 35 have not been previously observed (Table 2).TABLE 2 New mutations in the ATM gene detected by the SSCP technique.Codon codon Mutation Localization change number Conseque 10744A > G*Promoter — — ↓-express ? 11482G > A* Exon 1b — — ↓-express ? IVS3 − IVS3 — — Disrupts 558A > T promoter +1 ISS 146C > G Exon 5 S > C 49Missense 381de1A Exon 7 T > X 127 FS, Term IVS8 − 3delGT Intron 8 — —Exon 9 skipped? 1028delAAA Exon 10 E > X 343 FS, Term A 1120C > T Exon11 Q > X 374 Term 1930ins16 Exon 15 S > X 644 FS, Term IVS16 + 2T > CIntron 16 — — Ex. 16 skipped? 2572T > C Exon 19 F > L 858 MissenseIVS21 + IG > A Intron 21 — — Ex. 21 skipped? 3085delA Exon 23 T > X 1029FS, Term 3381delTCA Exon 25 Q > X 1127 FS, Term G 3602delTT Exon 27 F >X 1200 FS, Term 4052delT Exon 29 L > X 1351 FS, Term 4396C > T Exon 31R > X 1466 Term 5188C > T Exon 37 R > X 1730 Term 5290delC Exon 37 L > X1730 FS, Term 5546delT Exon 39 I > X 1849 FS, Term 5791G > CCT Exon 41D > X 1931 FS, Term 6047A > G Exon 43 D > G 2016 Missense IVS44 − 1G > TIntron 44 — — Ex. 45 skipped? 6672delGG/ Exon 48 M − A − L − 2224In-frame 6677delTAC R > I − S deletion G 6736del11/ Exon 48 C − I − K −2246 In-frame 6749del7 D − I − L − deletion T > H 7159insAG Exon 51 F >X 2387 FS, Term CC 7671delGTT Exon 54 L > X 2257? FS, Term T 7705del4Exon 54 D > X 2569? FS, Term 7865C > T Exon 55 A > V 2621 New spl donor7989deITGT Exon 56 — 2663 Val deleted 8177C > T Exon 58 A > V 2726Missense 8545C > T Exon 60 R > X 2849 Term 8565T > A Exon 60 S > R 2855Missense IVS64 + 1G > Intron 64 — — Ex. 64 T skipped? 9010del28 Exon 65K > X 3004 FS, Term

[0212] Most of the new mutations corresponded to protein truncatingmutations, as previously reported (Telatar et al., (1996), supra; P.Concannon & R. A. Gatti, “Diversity of ATM Gene Mutations in Patientswith Ataxia-Telangiectasia,” Hum. Mutat. 10:100-107 (1997); Telatar etal.(1998a), supra). These had not been detected before because RNA wasnot available for PTT testing. Mutations causing splicing defects werealso found (IVS8-3delGT, IVS16+2T>C, IVS21+G>A, IVS44-1G>T, 7865C>T,IVS64+1G>T), along with missense mutations, some of the latter affectingamino acids that are conserved in both mouse and pufferfish genomes(146C>T, 6047A>G, 8177C>T, 8565T>A); one of them was not conserved(2572T>C). It is interesting to note two in-frame deletions found in twounrelated A-T patients, both homozygous, in exon 48. These complexdeletions affected amino acids that are again conserved in the mouse andpufferfish genomes. Since the untranslated and promoter regions of theATM gene were included in the SSCP screening, the experiments also wereable to detect DNA changes in those areas. In the promoter region,detected 10744A>G and 11482G>A were detected in two unrelated compoundheterozygotes (i.e., A-T patients); each of these DNA changes couldaffect the normal expression of the gene. Experiments are in progress totest this possibility. A mutation was also detected at IVS3-558A>T,which disrupts the initiation start site (+1) for the putative promoterin intron 3 reported by Platzer et al. (1997), supra.

[0213] Mega-SSCP

[0214] An assay was subsequently developed that would analyze the entireATM gene for a small number of samples using only three SSCP gels andthe primers specified in Table 1, above. Mega-SSCP also includedsequential loading of sets of 2 or 3 PCR products, 29 sets of whichappear in FIG. 2.

[0215] ATM Polymorphisms and Rare Variants

[0216] The screening of the ATM gene detected 34 intragenicpolymorphisms or rare variants, all being new. Ten of these were commonpolymorphisms, with frequencies for the minor allele between 14-45%(Table 3). The experiments also detected and characterized 24 variantsthat were not common; the minor allele was found in less than 6% ofindividuals studied (Table 4). Since these tended to be found in otherunrelated A-T patients, rather than in the 40 normal unrelated controls,it is problematic as to whether to refer to these as “uncommonpolymorphisms” or “rare variants”. There is also a strong possibilitythat some of these changes may have functional effects on the ATM geneor protein. Some minor alleles were observed in <1% of individualstested, and these were considered as “rare variants”. The allelicfrequencies of common polymorphisms were characterized using 80independent normal chromosomes (CEPH families) (J. Dausset et al.,“Centre d'Etude du Polymorphisme Humaine (CEPH): Collaborative GeneticMapping of the Human Genome,” Genomics 6:575-577 (1990)). The allelicfrequencies of uncommon polymorphisms and rare variants were calculatedby including the 40 normal controls and 100 previously tested A-Tpatients (35 Turkish, 25 American, 20 Polish, 5 Costa Rican, 5 Spanishand 10 miscellaneous)—in all, 280 unrelated chromosomes. Any patientswho shared a minor allele of less than 6% frequency were alsohaplotyped; those with shared haplotypes were considered as related withfounder effect mutations and were included only once in calculating thefrequency of the minor allele. Because control populations shouldtheoretically be ethnically matched, but were not available in mostcases, the allelic frequencies of the reported uncommon polymorphismsand rare variants should be considered only as estimates. TABLE 3 Commonpolymorphisms in the ATM gene. Allelic frequency (N = 80) 10807A > G*72:28 IVS3 − 122T > C 55:45 IVS6 + 70delT 71:29 IVS16 − 34C > A 75:25IVS22 − 77T > C 72:28 IVS24 − 9delT 86:14 IVS25 − 13delA 63:37 5557G > A75:25 IVS48 − 69insATT 61:39 IVS62 − 55T > C 69:31

[0217] TABLE 4 Uncommon polymorphisms (>1%) and rare variants (<1%) inthe ATM gene. Estimated allelic frequency (%) (N = 280) 10677G > C* 110742G > T* 0.5 10819G > T* 0.5 10948A > G* 1 IVS3 − 300G > A 4 IVS8 −24del5 1 IVS13 − 137T > C 1 IVS14 − 55T > G 5 1986T > C 0.5 IVS20 +27delT 1 IVS23 − T61 > C 0 IVS25 − 35T > A 3 IVS27 − 65T > C 2 IVS30 −54T > C 0.5 4362A > C 0.5 IVS38 − 8T > C 6 5793T > C 1 IVS47 − 11G > T0.5 IVS49 − 16T > A 0.5 IVS53 + 34insA 1 IVS60 − 50deITTAGTT 0.5 IVS62 +8A > C 2 IVS62 − 65G > A 0.5 9200C > G 1.5

[0218] Discussion

[0219] Since PTT only detects mutations resulting in prematuretermination of the ATM protein (Telatar et al. (1996), supra) andrequires RNA, a need still exists for using sequence-basedmethodologies, such as SSCP, in searching for ATM mutations using DNA asstarting template. However, most of these technologies are verycumbersome due to the size of the ATM gene. Approaches like themega-SSCP that are described herein provide a workable solution for thepresent.

[0220] As previously reported, 70% of mutations in 48 patients weredetected by PTT when RNA was used as template for studying only thecoding region. In the present study, RNA was not available from mostpatients; DNA was the only material available. In addition, this studyconstitutes the first screening of the ATM gene in A-T homozygotes thatincludes the coding region, flanking intronic areas, and promoterregions. This approach permitted the detection of missense andexpression regulation mutations, which would not have been detected byPTT screening. Most strikingly, however, many more DNA changes werefound in intronic sequences.

[0221] In A-T patients, mutations have been detected throughout the ATMgene, without evidence of a mutational hot spot. Furthermore, when adatabase generated from the work reported herein is added to all otherreported mutations (Concannon and Gatti (1997), supra;http://www.vmmc.org/vmrc/atm.htm), there is no ATM exon without amutation.

[0222] Although the SSCP conditions were carefully optimized to detectall previously known mutations in a PCR fragment, we achieved adetection efficiency of only 70% in these experiments. SSCP has beenwidely used to screen for mutations. Grompe (M. Grompe, “The RapidDetection of Known Mutations in Nucleic Acids,” Nature Genet.5:111-117(1993)) claimed an efficiency of 100%. The data reported hereindo not support this claim, at least when screening for a large varietyof unknown mutations in a large gene like ATM.

[0223] Many abnormal SSCP patterns, when sequenced, were not mutationsat all but corresponded to: (1) common polymorphisms (i.e., with a minorallele of >14%) that had been previously observed (unpublished), 2)uncommon polymorphisms (i.e., with a minor allele of <6%), or 3) rarevariants (i.e., with a minor allele of <1%). So far, there have beenonly two reports about polymorphisms in the ATM gene (I. Vorechovsky etal., “ATM Mutations in Cancer Families,” Cancer Res. 56: 4130-4133(1996); T. Dork et al., “A Frequent Polymorphism of the Gene Mutated inAtaxia-Telangiectasia,” Mol. Cell. Probes 11:71-73 (1997)). A total of34 new ATM polymorphisms or rare variants were identified. Most of them(29) were found outside the coding regions, in either intronic sequencesor untranslated regions (UTR). Ten of them had minor allele frequenciesof >14%, and were identified as “common”. The remaining 24 had minorallele frequencies of <6%. These can be grouped as either uncommonpolymorphisms or rare variants; they are very uncommon in normalpopulations (Vorechovsky et al. (1996), supra). However, it isproblematic whether some of these changes are new “polymorphisms” thathave not yet had time to become fixed into the general population or,alternatively, are changes that represent functional mutations ofregulatory sequences. Most were located in flanking intronic sequences,away of the exon/intron boundaries. In some cases, these changes in theATM sequence could have phenotypic effects, as has already beensuggested for the IVS40+1126A>G mutation (C. McConville et al.,“Mutations Associated with Variant Phenotypes in Ataxia-Telangiectasia,”Am. J. Hum. Genet. 59:320-330 (1996)).

[0224] In the few situations where RNA was available to test theconsequences of a particular DNA change, no abnormalities were noted.However, it is still possible that these changes could have a phenotypiceffect by reducing the normal level of RNA expression. That thesechanges have only been detected in A-T populations, and not in normalcontrol samples, is disturbing. Such variants/polymorphisms have beenpreviously described for other monogenic diseases; in cystic fibrosis(M. Chillon et al., “Mutations in the Cystic Fibrosis Gene in Patientswith Congenital Absence of the Vas Deferens,” N. Engl. J. Med.332:1475-1480 (1995); X. Estivill, “Complexity in a Monogenic Disease,”Nature Genet. 12:350 (1996)) and in adenomatous polyposis coli (S.Pedemonte et al., “Novel Germline APC Variants in Patients with MultipleAdenomas,” Genes Chromosomes Cancer 22:257-267 (1998)) such sequencechanges are associated with an altered phenotype that depends upon theratio of expression of the normal/abnormal message. These mutationswould have been initially classified as“silent DNA variants”. Makridakiset al. (N. Makridakis et al., “A Prevalent Missense Substitution thatModulates Activity of Prostatic Steroid 5-Reductase,” Cancer Res.57:1020-1022 (1997)) have similarly observed modulated enzymaticactivity with missense substitutions in the prostatic steroid5-reductase gene. This also could be the situation for some of the rarevariants that have been detected that are unique to the A-T population.Studies are underway to test this hypothesis.

[0225] Identifying new mutations is contributing valuable informationfor genetic counseling, prenatal testing, carrier detection, and fordesigning rapid assays for specific founder effect mutations. As aresult of previous studies, six rapid DNA assays can now be used todetect >99% of all Costa Rican ATM mutations (Telatar et al. (1998b),supra). These mutations should also provide indirect information aboutfunctional domains of the ATM molecule, and help to clarify the role ofthe ATM gene in cancer predisposition. Characterization of newpolymorphisms within the ATM gene should be useful as a marker system inthe genetic diagnosis of A-T family members, in construction ofhaplotypes, and in loss of heterozygosity (LOH) studies. Knowing thesemutations should also prove useful in designing chip arrays forautomated mutation detection.

Advantages of the Present Invention

[0226] The present invention provides a novel method for screeninglarge, polyexonic genes for mutations and polymorphisms, known asmega-SSCP. The mega-SSCP method provides a method for screening genesfor multiple polymorphisms and mutations at once by using a small numberof electrophoreses. This method is of particular use for detection ofmutations and polymorphisms in the ATM gene, but can be used for manyother genes as well. The method is particularly useful for large,polyexonic, eukaryotic genes, particularly those where mutations orpolymorphisms can occur at many points within the gene and not merely atone or a few hot spots.

[0227] The present invention also provides novel mutations andpolymorphisms in the ATM gene that are useful in screening DNA. Inparticular, the novel polymorphisms are important because it isnecessary to screen out such polymorphisms in screening for mutationsthat cause a loss of function of the ATM gene and thus increase thesignal-to-noise ratio for screening of DNA for such mutations.

[0228] Although the present invention has been described in considerabledetail, with reference to certain preferred versions thereof, otherversions and embodiments are possible. Therefore, the scope of theinvention is determined by the following claims.

We claim:
 1. An isolated and purified nucleic acid fragment comprisingnucleic acid having complementarity or identity to a mutation in theataxia-telangiectasia mutated (ATM) gene, the mutation being selectedfrom the group consisting of: (a) 10744A>G; (b) 11482G>A; (c)IVS3-558A>T; (d) 146C>G; (e) 381delA; (f) IVS8-3delGT; (g) 1028delAAAA;(h) 1120C>T; (i) 1930ins16; (j) IVS16+2T>C; (k) 2572T>C; (l) IVS21+1G>A;(m) 3085delA; (n) 3381delTGAC; (o) 3602delTT; (p) 4052delT; (q) 4396C>T;(r) 5188C>T; (s) 5290delC; (t) 5546delT; (u) 5791G>CCT; (v) 6047A>G; (w)IVS44-1G>T; (x) 6672delGC/6677delTACG; (y) 6736dell 1/6749del7; (z)7159insAGCC; (aa) 7671delGTTT; (ab) 7705del14; (ac) 7865C>T; (ad)7979delTGT; (ae) 8177C>T; (af) 8545C>T; (ag) 8565T>A; (ah) IVS64+1G>T;and (ai) 9010del28.
 2. The fragment of claim 1 wherein the fragment hascomplementarity to the mutation in the ATM gene, is hairpin shaped, iscovalently linked to a fluorophore and to a quencher, and has astructure such that the fluorophore is internally quenched by thequencher when the fragment is not base-paired and such that the internalquenching is relieved when the fragment is base-paired, therebyrestoring fluorescence of the fluorophore.
 3. The fragment of claim 1wherein the fragment is DNA, has complementarity to the mutation in theATM gene, and wherein the fragment further includes, covalently linkedto either its 5′-end or to its 3′-end, a segment of about 40 bases, thesegment of about 40 bases comprising a repeating unit of dCdG or dGdC.4. A method for testing a DNA sample of a human for the presence orabsence of a mutation in the ATM gene comprising the steps of: (a)providing a sample of DNA from a human; and (b) testing the sample forthe presence of a mutation in the ATM gene, the mutation being selectedfrom the group consisting of: (i) 10744A>g; (ii) 11482G>A; (iii)IVS3-558A>T; (iv) 146C>G; (v) 381delA; (vi) IVS8-3delGT; (vii)1028delAAAA; (viii) 1120C>T; (ix) 1930ins16; (x) IVS16+2T>C; (xi)2572T>C; (xii) IVS21+1G>A; (xiii) 3085delA; (xiv) 3381delTGAC; (xv)3602delTT; (xvi) 4052delT; (xvii) 4396C>T; (xviii) 5188C>T; (xix)5290delC; (xx) 5546delT; (xxi) 5791G>CCT; (xxii) 6047A>G; (xxiii)IVS44-1G>T; (xxiv) 6672delGC/6677delTACG; (xxv) 6736dell 1/6749del7;(xxvi) 7159insAGCC; (xxvii) 7671delGTTT; (xxviii) 7705del14; (xxix)7865C>T; (xxx) 7979delTGT; (xxxi) 8177C>T; (xxxii) 8545C>T; (xxxiii)8565T>A; (xxxiv) IVS64+1G>T; and (xxxv) 9010del28.
 5. An isolated andpurified nucleic acid fragment comprising nucleic acid havingcomplementarity or identity to a polymorphism in theataxia-telangiectasia mutated (ATM) gene, the polymorphism beingselected from the group consisting of: (a) 10807A>G; (b) IVS3-122T>C;(c) IVS6+70delT; (d) IVS16-34C>A; (e) IVS22-77T>C; (f) IVS24-9delT; (g)IVS25-13delA; (h) 5557G>A; (i) IVS48-69insATT; and (j) IVS62-55T>C. 6.The fragment of claim 5 wherein the fragment has complementarity to thepolymorphism in the ATM gene, is hairpin shaped, is covalently linked toa fluorophore and to a quencher, and has a structure such that thefluorophore is internally quenched by the quencher when the fragment isnot base-paired and such that the internal quenching is relieved whenthe fragment is base-paired, thereby restoring fluorescence of thefluorophore.
 7. The fragment of claim 5 wherein the fragment is DNA, hascomplementarity to the polymorphism in the ATM gene, and wherein thefragment further includes, covalently linked to either its 5′-end or toits 3′-end, a segment of about 40 bases, the segment of about 40 basescomprising a repeating unit of dCdG or dGdC.
 8. A method for testing aDNA sample of a human for the presence or absence of a polymorphism inthe ATM gene comprising the steps of: (a) providing a sample of DNA froma human; and (b) testing the sample for the presence of a polymorphismin the ATM gene, the polymorphism being selected from the groupconsisting of: (i) 108-7A>G; (ii) IVS3-122T>C; (iii) IVS6+70delT; (iv)IVS16-34C>A; (v) IVS22-77T>C; (vi) IVS24-9delT; (vii) IVS25-13delA;(viii) 5557G>A; (ix) IVS48-69insATT; and (x) IVS62-55T>C.
 9. An isolatedand purified nucleic acid fragment comprising nucleic acid havingcomplementarity or identity to a polymorphism in theataxia-telangiectasia mutated (ATM) gene, the polymorphism beingselected from the group consisting of: (a) 10677G>C; (b) 10742G>T; (c)10819G>T; (d) 10948A>G; (e) IVS3-300G>A; (f) IVS8>24del5; (g)IVS13-137T>C; (h) IVS14-55T>G; (i) 1986T>C; (j) IVS20+27delT; (k)IVS23-76T>C; (l) IVS25-35T>A; (m) IVS27-65T>C; (n) IVS30-54T>C; (o)4362A>C; (p) IVS38-8T>C; (q) 5793T>C; (r) IVS47-11G>T; (s) IVS49-16T>A;(t) IVS53+34insA; (u) IVS60-50 delTTAGTT; (v) IVS62+8A>C; (w)IVS62-65G>A; and (x) 9200C>G.
 10. The fragment of claim 9 wherein thefragment has complementarity to the polymorphism in the ATM gene, ishairpin shaped, is covalently linked to a fluorophore and to a quencher,and has a structure such that the fluorophore is internally quenched bythe quencher when the fragment is not base-paired and such that theinternal quenching is relieved when the fragment is base-paired, therebyrestoring fluorescence of the fluorophore.
 11. The fragment of claim 9wherein the fragment is DNA, has complementarity to the polymorphism inthe ATM gene, and wherein the fragment further includes, covalentlylinked to either its 5′-end or to its 3′-end, a segment of about 40bases, the segment of about 40 bases comprising a repeating unit of dCdGor dGdC.
 12. A method for testing a DNA sample of a human for thepresence or absence of a polymorphism in the ATM gene comprising thesteps of: (a) providing a sample of DNA from a human; and (b) testingthe sample for the presence of a polymorphism in the ATM gene, thepolymorphism being selected from the group consisting of: (i) 10677G>C;(ii) 10742G>T; (iii) 10819G>T; (iv) 10948A>G; (v) IVS3-300G>A; (vi)IVS8>24del5; (vii) IVS13-137T>C; (viii) IVS14-55T>G; (ix) 1986T>C; (x)IVS20+27delT; (xi) IVS23-76T>C; (xii) IVS25-35T>A; (xiii) IVS27-65T>C;(xiv) IVS30-54T>C; (xv) 4362A>C; (xvi) IVS38-8T>C; (xvii) 5793T>C;(xviii) IVS47-11G>T; (xix) IVS49-16T>A; (xx) IVS53+34insA; (xxi)IVS60-50 delTTAGTT; (xxii) IVS62+8A>C; (xxiii) IVS62-65G>A; and (xxiv)9200C>G.